Not applicable.
Not applicable.
In microscopy, and particularly in fluorescence correlation spectroscopy (FCS), as for example is known from the ConfoCor of Carl Zeiss, the time series of signals produced by individual fluorescence events and detected by so-called Single Photon Counting are, for evaluation, either time-correlated with themselves (autocorrelation) or are time-correlated with the time series of signals of a second input channel (cross-correlation). The correlation evaluation takes place by means of special hardware correlators, such as are for example offered by the ALV-Laser Vertriebsgesellschaft company of Langen, Germany under the designation xe2x80x9c5000 Multiple Tau Correlatorxe2x80x9d. Such correlators operate according to the so-called multiple tau process, in which the input signals are respectively multiplied together over a correlation step time and the resulting products are added, the correlation step time being logarithmically extended stepwise. The advantage of this process is that the computation cost remains relatively small, even for long correlation times. It is however disadvantageous that a low-pass filtering takes place due to the combination of the input signals in the higher correlation steps. Moreover, the original data are lost, so that a processing and a subsequently following new or different evaluation is not possible. For the correlation evaluation of a single channel with individual pulse signals, and thus a signal sequence which gives binary data 0 and 1, where the 1 occurs only occasionally, it is known to sample the input channel at a fixed frequency and to record and store only the time intervals between the individual pulses. The correlation calculation then takes place simply, by the determination of all the pulse time intervals, which have appeared in the pulse sequence. An application of this process to the signals of several input channels is however not known.
The invention has as its object a process for the recording of pulse signals of several input channels, making possible as compact as possible a storage of the information without loss of information.
This object is attained by a process for the recording of pulse signals of at least two input channels, comprising:
sampling said input channels with a predetermined sampling frequency for events which have occurred, and
after detection of an event in at least one of said input channels, or after overflow of a counter, storing the present state of all said input channels in a memory register together with a magnitude characterizing a time interval to the last storage operation. This object also is attained by a process for the recording of pulse signals of at least two input channels, comprising:
sampling said input channels with a predetermined sampling frequency of events which have occurred, and
after detection of an event in at least one of the input channels, or after overflow of a counter, storing the states of all said input channels in the sampling cycle in which an event took place and for a predetermined number of sampling cycles after the occurrence of the first event, together with a magnitude characterizing the time interval to the last preceding storage operation.
In a first embodiment of the invention, the several input channels are sampled at a predetermined constant frequency for occurring events, and after the detection of an event in one of the input channels or after overflow of a counterxe2x80x94according to which of these two events occurs firstxe2x80x94the present state of all the input channels is stored in a memory register together with a magnitude characterizing the time interval from the last storage operation.
In a second embodiment of the invention, the several input channels are likewise sampled at a predetermined constant frequency for occurring events. And in this second embodiment also, the storage takes place after the detection of an event in one of the input channels or after the overflow of a counter, according to which of the two events occurs first. However, in this embodiment, the states of the input channels in the sampling cycle in which the event occurred, and additionally, for a predetermined number of sampling cycles after the occurrence of the event, are stored, together with a magnitude characterizing the time interval to the last storage.
In both embodiments, the information concerning the signal sequences in all the input channels is completely retained in the stored data; by means of the time interval coding which was carried out, the raw data are present in a form which makes possible a later autocorrelation and/or cross-correlation evaluation of the histograms of the pulse time intervals of the input channels. The storage requirement is in both cases dependent, primarily on the frequency of the events occurring in the input channels, and only secondarily on the sampling frequency.
In the first embodiment, only a single bit is required in the respectively stored words for each input channel; the remaining bits of each word are available. for the representation of the time interval to the last storage operation. With two input channels and storage as 16-bit words, 14 bits thus result for the representation of the time interval. This embodiment makes possible an optimally efficient use of the storage space with signal sequences which have only very few events in relation to the duration of a single sampling cycle, so that in most cases a storage operation takes place because of the overflow of a counter. However, the rate of data to be stored becomes extremely high for signal sequences with many events, in which an event occurs in each sampling cycle.
In the second embodiment, there is required in each stored word for each input channel a number of bits which corresponds to the number of the predetermined sampling cycles over which the channel states are also stored, and in addition a further bit for the state in the scanning cycle which triggers the storage operation. With two input channels, a storage operation over respectively three sampling cycles following the first occurring event or the counter overflow, and storage as 16-bit words, consequently requires 8 bits for the storage of the states of the input channels, so that only a further 8 bits are available for the storage of the time interval to the last preceding storage. With signal sequences which have only very rare events, and consequently in most cases the storage operation is triggered by the overflow of a counter, the storage is inefficient in comparison with the first embodiment, since only a smaller number of bits is available for the counter, and a counter overflow results correspondingly more frequently. This disadvantage is however not very troublesome, since with signal sequences with rarely occurring events, the total storage requirement is small and therefore not critical. On the other hand, in comparison with the first embodiment, the storage space required with signal sequences with frequently occurring events, and hence also the maximum required storage space, is clearly reduced. Thus there result in the above numerical example and with a sampling rate of 20 MHz in the second embodiment, a maximum storage rate (when an event occurs in each sampling cycle) of 10 Mbyte/s, and a minimum storage rate (when no event occurs and the storage operation is consequently triggered by the counter overflow) of 155 kbyte/s. In contrast to this, the maximum data rate in the first embodiment and at the same sampling frequency is 20 Mbyte/s.
The data recorded according to the invention can be subsequently read out and/or processed. Furthermore the recorded data can also be subjected to a correlation evaluation, in which either the data of each individual channel are correlated with themselves (autocorrelation), or the data of two input channels are correlated together (cross-correlation). The stored data are therefore already in a form suitable for a linear correlation algorithm in which the correlation function is calculated without loss, that is, without any loss of information, from the histogram of the pulse intervals in time.
The combined application of two different correlation algorithms is however particularly advantageous, one of them, the linear algorithm, being applied for short correlation times and the second, the multiple tau process, being applied for longer correlation times. The boundary between the two algorithms, that is, the correlation time, which represents the boundary between the two algorithms, is then selectable by the user, by software implementation. The linear algorithm operates without loss and requires smaller computing capacity for short correlation times than does the multiple tau process; the required computing capacity then increases linearly with the correlation time. The computing capacity required for the multiple tau process is limited in comparison with this and is nearly independent of the correlation time. The boundary between the two processes is therefore appropriately set at such correlation times at which the required computing capacities for the two algorithms mutually correspond.
The process according to the invention is in particular best suited to data recording in fluorescence correlation spectroscopy, in which fluorescence signals from microscopically small volumes are recorded and are evaluated by correlation calculations. Correspondingly, the invention also preferably finds application in connection with confocal microscopes.